Large-scale consumption of the traditional, fossil fuels (petroleum-based fuels) in the last few decades has contributed to high cost and high levels of pollution. Moreover, the realization that the world stock of petroleum is not boundless, combined with the growing environmental awareness, has stimulated new initiatives to investigate the feasibility of alternative fuels such as cellulosic ethanol, which can reduce CO2 production.
Processes presently employed for the production of ethanol include the following operational phases: (a) fermentation of the appropriate raw materials to obtain fermented products and (b) distillation of the products obtained by fermentation whereby ethanol is produced. A yeast belonging to the genus Saccharomyces, Saccharomyces cerevisiae, has been mainly used as a seed strain for ethanol fermentation. Saccharomyces cerevisiae cells are round to ovoid, 5-10 micrometers in diameter and can efficiently utilizes hexose including glucose, mannose, galactose etc. The fermentation process mentioned above includes seeding Saccharomyces cerevisiae is a medium containing nitrogen source, carbon source and trace elements etc. and performing fermentation under appropriate conditions, and it involve the chemical reaction: C6H12O6→2 CH3CH2OH+2 CO2.
Various sources of biomass can be used for alcohol production. The many and varied raw materials used in the manufacture of ethanol via fermentation are conveniently classified under three types of agricultural raw materials: sugar, starches, and lignocellulose materials. Although biomass-derived ethanol may be produced by fermentation of sugars or starches that are obtained from many different sources, so far, however, the substrates for industrial scale production or fuel alcohol are cane sugar and corn starch. The techniques of using sugars or starches to produce ethanol are well-developed; however, these substrates are of the high costs, it may take away from food supply and the production of ethanol from these sources is insufficient in meeting future demands for fuel industry. Therefore, there is a growing interest in producing ethanol from lignocelluloses. Lignocelluloses is a desirable alternative over other ethanol feedstocks such as corn grain since it is renewable, abundant, does not take away from the food supply and is available at a relatively low cost. Expanding fuel ethanol production requires the ability to use lower-cost feedstocks. Presently, only lignocellulosic feedstock from plant biomass would be available in sufficient quantities to substitute the crops used for ethanol production. The major fermentable sugars from lignocellulosic materials are glucose and xylose, constituting respectively about 40% and 25% of lignocellulose.
However, most yeasts that are capable of alcoholic fermentation, like Saccharomyces cerevisiae, are not capable of using xylose as a carbon source. To enable the commercial production of ethanol from lignocellulose hydrolysate, an organism possessing both these properties would be required. Scientists utilize genetic technology or strain taming to improve fermentation of xylose by yeasts or bacteria for the production of ethanol. U.S. Pat. No. 5,789,210 provides yeast strains capable of effectively fermenting xylose alone or simultaneously with glucose can be created using recombinant DNA and gene cloning techniques and these techniques have been used to create recombinant yeasts containing cloned xylose reductase (XR), xylitol dehydrogenase (XD), and xylulokinase (XK) genes which are fused to promotors not inhibited by the presence of glucose. U.S. Pat. No. 6,582,944 relates to new recombinant yeast strains transformed with xylose reductase and/or xylitol dehydrogenase enzyme genes, which is capable of reducing xylose to xylitol and consequently of producing xylitol in vivo. Bjorn et al. provide a yeast strain, TMB 3001, by transforming it with xylase reductase and/or xylitol dehydrogenase enzyme genes to solve the problem of being unable to metabolism xylose to produce ethanol (Biorn J, Barbel H H. The non-oxidative pentose phosphate pathway controls the fermentation rate of xylose but not of xylose in TMB 3001, 2002, FEMS Yeast Research 2:227-282). However, it still has the following problems: low xylose consumption rate (0.13 g xylose/g biomass/hour) and low ethanol yield (0.15 g product/g consumed xylose). To solve these problems, Johansson et al. further transform transaldolase gene to Saccharomyces cerevisiae to obtain a new strain, TMB 3026, which can increase xylose consumption rate from 0.12 to 0.23. (Biorn J, Barbel H H. The non-oxidative pentose phosphate pathway controls the fermentation rate of xylose but not of xylose in TMB 3001, 2002, FEMS Yeast Research 2:227-282); however, the requirements for industrial production still have not been met. Kaisa et al. further create a transformed yeast, TMB 3057, having an improved gene expression in xylose reductase and/or xylitol dehydrogenase and a deleted aldose reductase gene (GR3) to elevate ethanol yield and reduce formation of xylitol by-product (Kaisa K, Romain F, Barbel H H, Marie G G High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae, 2007, Appl. Microbiol. Biotechnol. 73: 1039-1046). However, this strain still has the problems of low xylose consumption rate (0.25 g xylose/g biomass/hour and low ethanol yield (0.27 g product/g consumed xylose). Furthermore, Elizebath et al. use gene modified yeast 422A (LNH-ST) that is transformed with xylose reductase gene of N crasser and C. parapsilosis and xylitol dehydrogenase gene of P. stipitis and has optimized codons of three amino acids (Eliabeth C, Miroslav S., Nancy W Y H, Nathan S M, Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae, 2010, FEMS Yeast Res. 10:385-393). This stain also utilizes GAPDH promoter to control pentose phosphate pathway of TKL1, TAL1, RKL1 and RPE1. However, it still has the following problems: low xylose consumption rate (0.27 g xylose/g biomass/hour at pH5) and low ethanol yield (0.785 g product/g consumed xylose) and low tolerance to acetic acid (the xylose consumption rate reduces to 015 from 0.354 when the medium contains 1 g/L acetic acid).
Therefore, there is a significant need in the art for a strain that provide for improved biomass (such as xylose) conversion to ethanol and a method for production of ethanol in higher yield.